Viruses alter cellular pathways in order to create an environment conducive for the infectious cycle, and to inactivate or escape cellular and systemic anti-viral defense systems. Faced with these challenges, viruses encode proteins or small RNAs to interact with key viral and cellular targets that normally regulate cell growth, death, transcriptional status and communication with systemic anti-viral defenses. DNA tumor viruses, such as simian virus 40 (SV40) have proved to be powerful tools for uncovering pathways that mediate cell proliferation, differentiation, and death. These viruses encode dominantly acting proteins that alter key cellular signal transduction pathways or regulate viral and cellular gene expression. The multifunctional large T antigen encoded by SV40 plays roles in both productive infection and tumorigenesis. T antigen drives cells into S phase by inhibiting the Rb-family of tumor suppressor proteins, and thus activating the expression of genes regulated by the E2F-family of transcription factors. This action requires a functional LXCXE motif that directs the association of T antigen with Rb-E2F complexes, and a J domain, that recruits hsc70. Simultaneously, T antigen binds the tumor suppressor p53 and inhibiting its transcriptional activation activity, and thus blocking p53-dependent cell-cycle arrest and apoptosis. Two key questions are whether the J domain and LXCXE motif of T antigen are sufficient to induce E2F-dependent transcription, and whether the inactivation of Rb and p53 tumor suppressors is the only T antigen functions that contribute to transformation. The regulation of gene expression also plays a role in productive infection. SV40 uses two mechanisms to downregulate early region transcription. First, T antigen binds to the early viral promoter directly inhibiting transcription. In addition, SV40 encodes miRNAs that bind to early mRNA and lower the expression of the viral tumor antigens. Consequently, SV40-infected cells are less susceptible to killing by cytotoxic T cells. In this application we propose to explore the roles played by SV40 control of gene expression in transformation and infection. Specifically we will: 1. determine whether the J domain and LXCXE motif sufficient for T antigen regulation of Rb-E2F interactions. 2. Assess the role of Rb- and p53-independent mechanisms in SV40-mediated transformation. 3. Distinguish the relative roles of the T antigen HR domain and the viral miRNAs in SV40 infection. 4. Determine whether T antigen autoregulation contribute to CTL evasion?